EP2658569B1 - Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh - Google Patents

Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh Download PDF

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EP2658569B1
EP2658569B1 EP11808600.8A EP11808600A EP2658569B1 EP 2658569 B1 EP2658569 B1 EP 2658569B1 EP 11808600 A EP11808600 A EP 11808600A EP 2658569 B1 EP2658569 B1 EP 2658569B1
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composition
inhibitor
antigen
mhc class
cells
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EP2658569A1 (fr
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Christina Lorenz
Mariola Fotin-Mleczek
Karl-Josef Kallen
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Curevac SE
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Curevac AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides

Definitions

  • the present invention relates to a composition
  • a composition comprising as a vaccine at least one tumour antigen and as an inhibitor at least one inhibitor of the major histocompatibility complex (MHC) class I restricted antigen presentation.
  • MHC major histocompatibility complex
  • Such an inhibitor can induce expression of T cell epitopes associated with impaired peptide processing (TEIPPs) in antigen presenting cells (APCs) and thereby stimulates specific T cell mediated immune responses against cancer which present these TEIPPs due to impairment of MHC class I restricted antigen presentation.
  • the inventive composition is therefore particularly suitable in the treatment of tumour diseases which are associated with loss of MHC class I restricted antigen presentation.
  • the present invention relates to the use of the composition as a medicament or for vaccination of a mammal.
  • the present invention also provides kit of parts comprising distinct compositions by two parts, e.g. for prior, concurrent or subsequent administration of the different parts.
  • an antigen is typically recognized by two distinct sets of highly variable receptor molecules, the immunoglobins, that serve as antigen receptors on B cells, and the antigen specific receptors of T cells.
  • the immunoglobins that serve as antigen receptors on B cells
  • the antigen specific receptors of T cells In this context peptides from the cytosol are usually bound to MHC class I molecules and recognized by CD8 + T cells, whereas peptides generated in vesicles are typically bound to MHC class II molecules and recognized by CD4 + T cells.
  • MHC class I molecules are expressed on all nucleated cells, whereas MHC class II molecules are preferentially expressed on professional antigen presenting cells.
  • One main object of the immune system is to distinguish in self and non-self antigens but also into infected cells or abnormal cells.
  • proteins occurring in and being produced by normal cells are continually degraded and replaced with newly synthesized proteins.
  • much cytosolic protein degradation is carried out by proteasomes.
  • proteasomes break the proteins into short peptides which are subsequently loaded on MHC class I molecules in the endoplasmatic reticulum (ER) by transporters associated with antigen processing (TAP).
  • TAPs are responsible for the delivery of the cytosolic peptide repertoire resulting from proteolytic breakdown of intracellular proteins into the endoplasmic reticulum (ER), thereby making these short peptides available for loading onto MHC class I molecules.
  • a second component of the newly formed complex is the TAP associated protein tapasin, which forms a bridge between MHC class I molecules and TAP1 and TAP2 and allows the transport of a degraded short peptide from the cytosol.
  • This complex is termed peptide loading complex (PLC). After binding the degraded short peptide the MHC class I molecule leaves the ER and is transported to the cell surface where it can be recognized by cytotoxic CD8 + T cells (see also Parcej and Tamé, Nature Chemical Biology 6, 572-580 (2010 )).
  • MHC class I epitope repertoire of normal cells does normally not induce the elimination of a cell, because the immune system has learned only to eliminate infected cells or abnormal cells with a divergent MHC class I epitope repertoire. Therefore CD8 + T cells only eliminate cells which bear the epitopes on MHC class I molecules fitting in their T cell receptor and which are recognized as foreign or abnormal from the immune system. In case of an infection with an intracellular pathogen many MHC class I molecules of the cell are filled with peptides of the pathogen instead of peptides from the normal cell and thus display a different epitope repertoire on MHC class I molecules.
  • tumour cells show a different epitope repertoire on MHC class I molecules when compared to normal cells, at least with regard to the quantity of one or more presented epitopes. Therefore, the immune system is able to specifically recognize abnormal cells (e.g. tumour cells) or infected cells by recognition of the epitopes on MHC class I molecules.
  • abnormal cells e.g. tumour cells
  • MHC class I molecules Since presentation of pathogenic peptides by MHC class I molecules signals CD8 + T cells to kill the infected cell, some pathogens, in particular viruses, have evolved ways of evading recognition by preventing the appearance of peptide:MHC class I complexes at the cell surface. For instance, the herpex simplex virus prevents the transport of viral peptides into the ER by producing a protein that binds to and inhibits TAP. Meanwhile, further mechanisms have been uncovered and it appears that many steps of antigen presenting on MHC class I molecules can be inhibited to prevent recognition of infected cells by CD8 + T cells. Such steps which can be inhibited in antigen presentation on MHC class I molecules include e.g.:
  • MHC class I abnormalities have been found in solid tumours of distinct origin, but also in hematopoietic diseases. These include structural alterations such as total, haplotype and allelic loss of the MHC class I heavy chain, deletions and point mutations, in particular in ⁇ 2-microglobulin and TAP1 as well as dysregulation of various components of the MHC class I antigen processing machinery (APM), which could occur at the epigenetic, transcriptional and posttranscriptional level (reviewed e.g. in Seliger, B.
  • APM antigen processing machinery
  • TAP-/- tumour cells display epitopes on MHC class I molecules which result from presentation via a TAP-independent pathway and mainly result from housekeeping genes ( van Hall, T., E. Z. Wolpert, et al. (2006), "Selective cytotoxic T-lymphocyte targeting of tumour immune escape variants.” Nat Med 12(4): 417-24 .). These epitopes were named TEIPPs (T-cell epitopes associated with impaired peptide processing). Previously, Wolpert et al.
  • Van Hall et al (2006) could show that these newly discovered epitopes can be exploited for immune intervention against processing-deficient tumours through adoptive T-cell transfer or peptide vaccination. They also suggested that in TAP-proficient tumour cells TEIPPs are out-competed by epitopes resulting from TAP-dependent presentation and therefore are not displayed in early-stage cancers. Moreover, they are not presented in normal "healthy" tissue, in which the TAP-machinery usually is functional. These peptides, although derived from self-antigens are not presented by normal cells. This explains why they act as immunogenic neoantigens.
  • TEIPPs are presented by Qa-1b or by its human homologue human leukocyte antigen (HLA)-E, respectively, which are conserved MHC class I like molecules, often categorized as "non-classical” or class 1b MHC ( Oliveira, C. C., P. A. van Veelen, et al. "The nonpolymorphic MHC Qa-1b mediates CD8+ T cell surveillance of antigen-processing defects.” J Exp Med, 2010, 207(1): 207-21 ). Similar to the classical MHC molecules, they accommodate small peptides in their binding grooves and present these on the cell surface.
  • HLA human leukocyte antigen
  • T cell receptor recognition of these non-classical MHCs has been described in the context of immunity against intracellular pathogens, e.g., Listeria, Salmonella, and Mycobacterium tuberculosi. Oliveira et al. (2010) furthermore described the existence of a surprisingly broad peptide repertoire that is presented by Qa-1b on cells with impairments in the antigen-processing machinery. These peptides are targeted by a unique population of CD8 + cytotoxic T cells. Normal cells with intact processing machinery were not recognized by these Qa-1b restricted CTLs, but partial defects readily resulted in the appearance of the immunogenic self-peptides, which are derived from housekeeping proteins.
  • pathogens particularly viruses
  • This function allows the pathogen to persist in the host for a long time without being discovered and eliminated by the immune system.
  • T cell epitopes associated with impaired peptide processing TEIPPs
  • tumour escape is often associated with loss in normal antigen presentation on MHC class I molecules.
  • CTLs cytotoxic T cells
  • TEIPPs T cell epitopes associated with impaired peptide processing
  • the present invention as defined by claim 1 solves the above object by a composition comprising an mRNA encoding at least one tumour antigen (herein: "vaccine component”) and at least one inhibitor of the major histocompatibility complex (MHC) class I restricted antigen presentation (herein: “inhibitor”), as well as by its use in methods of treatments, compositions for use in cancer therapy as defined by claim 10 and kits involving parts as defined by claim 15.
  • vaccine component at least one tumour antigen
  • MHC major histocompatibility complex
  • inhibitor inhibitor of the major histocompatibility complex
  • the vaccine and the inhibitor may be formulated together as a composition or separately by different compositions for use or as parts of a kit.
  • the term "vaccine/inhibitor combination” preferably means combined occurrence of a vaccine comprising an mRNA coding for at least one tumour antigen and of at least one inhibitor of MHC class I restricted antigen presentation (e.g. by two parts of the inventive kit or by the inventive composition), preferably within one treatment.
  • administration of the vaccine and the inhibitor may occur either simultaneously or timely staggered, either at the same site of administration or at different sites of administration, as further outlined below.
  • Such a vaccine/inhibitor combination may, on the one hand side, induce an active immune response and, on the other hand side, induce expression of T cell epitopes associated with impaired peptide processing (TEIPPs) via the inhibitor of the major histocompatibility complex (MHC) class I restricted antigen presentation.
  • TEIPPs impaired peptide processing
  • MHC major histocompatibility complex
  • the vaccine/inhibitor combination is thus suitable to effectively stimulate specific T cell mediated immune responses against cancer. More precisely, the vaccine/inhibitor combination is particularly suitable in the treatment of tumour diseases, which are associated with loss of MHC class I restricted antigen presentation and to further improve the immune response against such tumour cells.
  • the vaccine/inhibitor combination preferably allows eliciting a CD8 + specific adaptive immune response in a patient to be treated, preferably a mammal, by using, e.g., as a first component a vaccine, which allows generation of an immune response targeting the tumour cells.
  • Addition of an inhibitor of MHC class I restricted antigen presentation then preferably induces the presentation of TEIPPs in antigen presenting cells (APCs), which can induce an elimination of those escaped tumour cells, which present the same TEIPPs as the induced APCs.
  • APCs antigen presenting cells
  • the present invention is based on the surprising finding that administration of a vaccine directed against a tumour antigen in combination with an inhibitor of MHC class I restricted antigen presentation could strongly decrease the harmful impact of a disease to be treated, e.g. the growth rate of a tumour.
  • the inventors surprisingly found that treatment with a vaccine comprising a tumour antigen in combination with an inhibitor of MHC class I restricted antigen presentation unexpectedly improved inhibition of tumour growth in a synergistic manner.
  • the present invention does not require the administration of fusion proteins comprising an MHC class I chain, e.g.
  • the present invention employs mRNA coding for tumour antigens, which - in a preferred embodiment - do not correspond to fusion proteins (combining the antigen artificially to another protein) and, in a further preferred embodiment, do not correspond to fusion proteins comprising an MHC class I (heavy) chain or ⁇ 2m fused to whatever other protein, e.g. an epitope, if an mRNA coding for a fusion protein shall be used according to the invention.
  • the present invention is preferably not directed to nucleic acids or vectors corresponding to such fusion proteins. Still further in another embodiment, the present invention does preferably not make use of an MHC class heavy chain or ⁇ 2m as components of the vaccine/inhibitor combination or composition (or nucleic acids or vectors coding for the MHC class I heavy chain or ⁇ 2m). Accordingly, in such a situation, the vaccine/inhibitor combination or composition does not include an MHC class I heavy chain or ⁇ 2m, be it as a protein or as a nucleic acid or a vector coding for the MHC class I heavy chain or ⁇ 2m.
  • the combination or composition achieves the formation and presentation of TEIPPs of the antigen component itself. Accordingly, the antigen(s) of the combination/composition is/are presented in the form of TEIPPs.
  • tumour cells which show normal antigen presentation on MHC class I cells
  • additional treatment with an inhibitor of MHC class I restricted antigen presentation generates antigen presenting cells (APCs), which present the same epitopes (TEIPPs) as the escaped tumour cells. Therefore, these TEIPP specific APCs can induce a CD8 + specific immune response by specifically eliminating the TEIPP presenting tumour cells.
  • APCs antigen presenting cells
  • the object underlying the present invention is solved by a composition of claim 1 comprising an mRNA coding for at least one tumour antigen (vaccine component), preferably to be administered to a patient in need thereof, in combination with an inhibitor of the major histocompatibility complex (MHC) class I restricted antigen presentation as defined by claim 1, representing a vaccine/inhibitor combination.
  • vaccine component preferably to be administered to a patient in need thereof
  • MHC major histocompatibility complex
  • That specific inventive composition ensures that formation of TEIPPs of the antigen(s) (as provided by the composition) is induced and, accordingly, these TEIPPs of the administered antigens are presented on competent immune cells of the vaccinated patient.
  • This inventive composition comprising the vaccine component and an inhibitor of MHC class I restricted antigen presentation shows an extremely advantageous inhibition of tumour growth which could not be expected from the prior art. It is particularly advantageous on the one hand side to induce an adaptive immune response against tumour cells by vaccination which in turn present TEIPPs on their surface to escape from the immune system and on the other hand side to generate TEIPP-specific APCs by administration of an inhibitor of MHC class I restricted antigen presentation.
  • composition B comprising an mRNA encoding at least one tumour antigen for use in cancer treatment in combination with another composition (composition comprising the at least one inhibitor), defined as composition B.
  • composition B comprising the inhibitor and the composition comprising the vaccine component are preferably administered separatedly in time (in a time-staggered manner) and/or are administered at different administration sites. This means that the composition comprising the vaccine component may be administered e.g. prior, concurrent or subsequent to the composition B comprising the inhibitor, or vice versa.
  • composition comprising the vaccine component and composition B comprising the inhibitor may be administered at different administration sites, or at the same administration site, preferably, when administered in a time staggered manner.
  • the composition comprising the vaccine component is to be administered firstly and the composition B is to be administered subsequently to the vaccine composition. This procedure ensures that the selection on TEIPP expressing cells has already taken place, even though concurrent administration or administration, wherein the inhibitor composition B is to be administered prior to the vaccine composition, may lead to the same or at least comparable results.
  • an inhibitor of MHC class I restricted antigen presentation is as defined by claim 1. It may be capable to impair antigen presentation via MHC class I molecules, e.g. inducing transcriptional down-regulation of MHC class I molecules, inducing mRNA degradation of MHC class I components, inducing degradation of the pre-peptide loading complex in the ER, inducing inhibition of the formation of the peptide-loading complex (PLC), inducing retention of MHC class I molecules within the ER and prevention of PLC interactions, inducing degradation of PLC (peptide loading complex) components, inducing shutting off the crucial supply of peptide, inducing transit of the MHC complex to the cell surface, inducing down-regulation of cell surface molecules which induce mis-sorting or enhanced internalization of the PLC complex, inducing inhibition of the proteasom, or inducing any further suitable mechanism known to a skilled person to impair antigen presentation via MHC class I molecules.
  • PLC peptide-loading complex
  • an inhibitor of MHC class I restricted antigen presentation includes inhibitors by shutting off the crucial supply of peptide and is selected from inhibitors inhibiting TAP (US6 of HCMV, ICP47 of herpes simplex virus, UL49.5 of varicelloviruses, and BNLF2a of Epstein-Barr virus (EBV)).
  • TAP US6 of HCMV
  • ICP47 of herpes simplex virus ICP47 of herpes simplex virus
  • UL49.5 varicelloviruses
  • BNLF2a Epstein-Barr virus
  • the inhibitor of MHC class I restricted antigen presentation as defined by claim 1 can be provided in form of a (recombinant) protein or as a nucleic acid coding for the inhibitor as defined herein for coding nucleic acids.
  • the protein inhibitor of TAP activity shall be provided as a nucleic acid or a vector
  • the vector is preferably not a mammalian cell-infecting virus vector and, more preferably, not a Sendai virus vector.
  • the MHC class 1 inhibitor is not provided in the form of a protein (or a nucleic acid or vector expressing a TAP activity inhibiting protein, respectively), but corresponds to other classes of compounds, e.g. small organic compounds having the function of inhibiting TAP activity.
  • a vaccine is a composition comprising one or more mRNA(s) encoding tumour antigenic substances (antigens) or agents suitable for administration into an organism, in particular into one or more cell(s) or tissue(s) of said organism.
  • Administration of antigens to a subject by an mRNA encoding the same or in any other form suitable as vaccine typically leads to activation of the immune system, which responds by production of antibodies to the respective antigens (humoral immunity) and particularly of immune cells, more precisely by production of cytotoxic T cells (cellular immunity by CD8 + T cells) directed against the respective encoded antigens.
  • the present invention is typically not directed to the administration of (transfected or modulated) cells to the patient.
  • the vaccine/inhibitor combination does not refer to the administration, or, more specifically, injection of (ex vivo) transfected or modulated cells, in particular does not refer to the administration of ex vivo transfected or modulated immune cells, such as dendritic cells (DC), e.g. DC transfected/transduced with a TAP inhibitor, such as a viral TAP inhibitor (e.g. a protein derived from gene UL49.5, which may occur in various viruses, e.g. in herpes virus, PRV or varicellovirus).
  • DC dendritic cells
  • TAP inhibitor such as a viral TAP inhibitor (e.g. a protein derived from gene UL49.5, which may occur in various viruses, e.g. in herpes virus, PRV or varicellovirus).
  • the combination or composition does preferably not comprise modulated or transfected cells, in particular no transfected or modulated immune cells (e.g. antigen presenting cells), more particularly no transfected or modulated DC.
  • the combination/composition does preferably not correspond to a DC-based vaccine, or, more generally, does preferably not correspond to a cell-based (e.g. antigen presenting cell-based) vaccine or, more generally, the combination/composition is preferably free of cells.
  • the combination/composition to be administered provides the vaccine component as an mRNA and other components preferably as mRNA and/or protein, more preferably as mRNA.
  • the term "antigen" refers to a substance which is recognized by the immune system and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies or antigen-specific T-cells as part of an adaptive immune response.
  • the first step of an adaptive immune response is typically the activation of naive antigen-specific T cells by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing.
  • the three cell types that can serve as antigen-presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses.
  • Tissue dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by infection to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents to express MHC class II molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important to induce T cells. The presentation of antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells.
  • T cells recognize an antigen by their T cell receptors. They do not recognize and bind the antigen directly, but recognize short peptide fragments instead, e.g. short peptide fragments of pathogens' protein antigens, which are bound to MHC molecules on the surfaces of other cells.
  • T cells fall into two major classes that have different effector functions.
  • the two classes are distinguished by the expression of the cell-surface proteins CD4 and CD8. These two types of T cells differ in the class of MHC molecule that they recognize.
  • the two classes of MHC molecules - MHC class I and MHC class II molecules - differ in their structure and expression pattern on tissues of the body.
  • CD4 + T cells bind to a MHC class II molecule and CD8 + T cells to a MHC class I molecule.
  • MHC class I and MHC class II molecules have distinct distributions among cells that reflect the different effector functions of the T cells that recognize them.
  • MHC class I molecules present peptides from pathogens, commonly viruses to CD8 + T cells, which differentiate into cytotoxic T cells that are specialized to kill any cell that they specifically recognize. Almost all cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next. But not only pathogenic peptides from viruses are presented by MHC class I molecules, also self-antigens like tumour antigens are presented by them. MHC class I molecules bind peptides from proteins degraded in the cytosol and transported in the endoplasmic reticulum. Thereby, MHC class I molecules on the surface of cells infected with viruses or other cytosolic pathogens display peptides from these pathogen.
  • CD8 + T cells that recognize MHC class I:peptide complexes are specialized to kill any cells displaying foreign peptides and so rid the body of cells infected with viruses and other cytosolic pathogens.
  • the main function of CD4 + T cells (CD4 + helper T cells) that recognize MHC class II molecules is to activate other effector cells of the immune system.
  • MHC class II molecules are normally found on B lymphocytes, dendritic cells, and macrophages, cells that participate in immune responses, but not on other tissue cells. Macrophages, for example, are activated to kill the intravesicular pathogens they harbour, and B cells to secrete immunoglobulins against foreign molecules.
  • MHC class II molecules are prevented from binding to peptides in the endoplasmic reticulum and thus MHC class II molecules bind peptides from proteins which are degraded in endosomes. They can capture peptides from pathogens that have entered the vesicular system of macrophages, or from antigens internalized by immature dendritic cells or the immunoglobulin receptors of B cells. Pathogens that accumulate in large numbers inside macrophage and dendritic cell vesicles tend to stimulate the differentiation of TH1 cells, whereas extracellular antigens tend to stimulate the production of TH2 cells.
  • TH1 cells activate the microbicidal properties of macrophages and induce B cells to make IgG antibodies that are very effective of opsonising extracellular pathogens for ingestion by phagocytic cells
  • TH2 cells initiate the humoral response by activating naive B cells to secrete IgM, and induce the production of weakly opsonising antibodes such as IgG1 and IgG3 (mouse) and IgG2 and IgG4 (human) as well as IgA and IgE (mouse and human).
  • antigens of the vaccine included in the herein defined combination of a vaccine and an inhibitor of MHC class I restricted antigen presentation comprise any mRNA coding for any (protein) tumour antigen, antigenic epitope or tumour antigenic peptide falling under the above definition.
  • tumour antigens are preferably located on the surface of the (tumour) cell. Tumour antigens may also be selected from proteins, which are overexpressed in tumour cells compared to a normal cell. Furthermore, tumour antigens also include antigens expressed in cells which are (were) not themselves (or originally not themselves) degenerated but are associated with the supposed tumour. Antigens which are connected with tumour-supplying vessels or (re)formation thereof, in particular those antigens which are associated with neovascularization, e.g. growth factors, such as VEGF, bFGF etc., are also included herein.
  • growth factors such as VEGF, bFGF etc.
  • Antigens connected with a tumour furthermore include antigens from cells or tissues, typically embedding the tumour. Further, some substances (usually proteins or peptides) are expressed in patients suffering (knowingly or not-knowingly) from a cancer disease and they occur in increased concentrations in the body fluids of said patients. These substances are also referred to as "tumour antigens", even though they are not antigens in the stringent meaning of an immune response inducing substance.
  • tumour antigens can be divided further into tumour-specific antigens (TSAs) and tumour-associated-antigens (TAAs).
  • TSAs can only be presented by tumour cells and are usually never presented by normal "healthy” cells. They typically result from a tumour specific mutation.
  • TAAs which are more common, are usually presented by both tumour and healthy cells.
  • TAAs which are more common, are usually presented by both tumour and healthy cells.
  • tumour antigens can also occur on the surface of the tumour in the form of, e.g., a mutated receptor. In this case, they can be recognized by antibodies.
  • tumour antigens are selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA
  • tumour antigens may preferably be selected from the group consisting of MAGE-A1 (e.g. MAGE-A1 according to accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g. MAGE-A6 according to accession number NM_005363), MAGE-C1, MAGE-C2, melan-A (e.g. melan-A according to accession number NM_005511), GP100 (e.g. GP100 according to accession number M77348), tyrosinase (e.g. tyrosinase according to accession number NM_000372), surviving (e.g.
  • CEA e.g. CEA according to accession number NM_004363
  • Her-2/neu e.g. Her-2/neu according to accession number M11730
  • WT1 e.g. WT1 according to accession number NM_0003708
  • PRAME e.g. PRAME according to accession number NM_006115
  • EGFRI epidermal growth factor receptor 1
  • MUC1 e.g. mucin-1 according to accession number NM_002456
  • SEC61 G e.g.
  • Antigens from pathogens associated with impaired antigen presentation on MHC class I molecules are also disclosed and include antigens from viruses like herpes-, retro-, flavi-, arena-, and polyomaviruses including simian virus 40 (SV40), the K virus of mice, and the JC and BK viruses of humans, lymphocytic choriomeningitis virus (LCMV), Epstein-Barr virus (EBV), murine leukemia virus (MuLV), mouse mammary tumour virus (MMTV), herpes simplex virus (HSV), human T-lymphotropic virus type 1 (HTLV-1), hepatitis B, hepatitis C virus (HCV), cytomegalovirus (CMV), human immunodeficiency virus (HIV), Kaposi's sarcoma-associated herpesvirus (KSHV), human papilloma virus, West Nile Virus, adenovirus, measles virus, Rubella virus, Varicella zoster, bo
  • Tumour antigens as defined herein which are encoded by the mRNA of the vaccine/inhibitor combination, furthermore comprise or consist of fragments comprising a T cell antigenic epitope of a tumour antigen of 6 to 20 contiguous amino acids thereof or tumour antigenic variants of these antigens, particularly of protein or peptide antigens, wherein the variants may share a sequence identity with one of the aforementioned antigens of at least 80%, preferably 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 96 or even 97, 98 or 99% over the whole length of the protein or peptide antigen or its encoding nucleic acid sequence.
  • a "fragment" in the context of the present invention is preferably to be understood as a truncated protein or its encoding nucleic acid, i.e. a protein which is N-terminally, C-terminally or intrasequentially truncated compared to the amino acid sequence of the original (wild type) protein or its encoding nucleic acid, respectively.
  • fragments consisting of or comprising an antigenic epitope or their encoding nucleic acids are preferred.
  • Fragments of such tumour antigens of the vaccine/inhibitor combination consist of or comprise a sequence having or encoding a length of 6 to 20 contiguous) amino acids of a (protein or peptide) antigen as defined above, e.g. fragments as processed and presented by MHC class I molecules, preferably consisting of or comprising a sequence having or encoding a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably consisting of or comprising a sequence having or encoding a length of about 13 or more amino acids, e.g.
  • T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form. These fragments are also termed as T cell epitopes.
  • Fragments of tumour antigens as defined herein of the vaccine component of the vaccine/inhibitor combination may also comprise or consist of B cell epitopes of those antigens.
  • B cell epitopes also called "antigen determinants" are typically fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably consisting of or comprising a sequence having or encoding 5 to 15 amino acids, more preferably consisting of or comprising a sequence having or encoding 5 to 12 amino acids, even more preferably consisting of or comprising a sequence having or encoding 6 to 9 amino acids, wherein the epitopes may be recognized by antibodies, i.e. in their native form.
  • tumour antigenic variants of the at least one tumour antigen of the vaccine component or the functional TAP inhibitor component of the vaccine/inhibitor combination, may comprise or consist of an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s), and exhibits a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90% and most preferably at least 97%, to the wild type amino acid sequence.
  • a nucleic acid coding for the inhibitor of MHC class I presentation as defined herein and as contained in the vaccine/inhibitor combination can be for instance a ssDNA, dsDNA, ssRNA, dsRNA, viral DNA, viral RNA, plasmid DNA or a messenger RNA (mRNA). It is particularly preferred that the inhibitor is encoded by an mRNA.
  • a messenger RNA is typically an RNA, which is composed of several structural elements, e.g. an optional 5'-UTR region, an upstream positioned ribosomal binding site followed by a coding region, an optional 3'-UTR region, which may be followed by a poly-A tail (and/or a poly-C-tail).
  • the (m)RNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more antigens or antigenic proteins or peptides or inhibitors of MHC class I restricted antigen presentation.
  • Such coding sequences in di-, or even multicistronic mRNAs may be separated by at least one IRES sequence, e.g. as defined herein.
  • the mRNA coding for the at least one tumour antigen or the nucleic acid molecule coding for the inhibitor as defined herein may be provided as a "stabilized nucleic acid", preferably as a stabilized RNA, more preferably as a RNA that is essentially resistant to in vivo degradation (e.g. by an exo- or endo-nuclease).
  • nucleic acid molecule as defined herein may contain nucleotide analogues/modifications e.g. backbone modifications, sugar modifications or base modifications.
  • a backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in inventive nucleic acid molecule as defined herein are chemically modified.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the inventive nucleic acid molecule as defined herein.
  • a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides of the nucleic acid molecule of the inventive nucleic acid molecule.
  • nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation.
  • the mRNA coding for the tumour antigen or the nucleic acid molecule coding for the inhibitor of the herein defined vaccine/inhibitor combination can contain a lipid modification.
  • the nucleic acid molecule coding for the antigen or the inhibitor of the herein defined vaccine/inhibitor combination may likewise be stabilized in order to prevent degradation of the nucleic acid molecule by various approaches, particularly, when RNA or mRNA is used as a nucleic acid molecule for the inventive purpose. It is known in the art that instability and (fast) degradation of RNA in general may represent a serious problem in the application of RNA based compositions. E.g., the terminal structure is typically of critical importance particularly for an mRNA.
  • cap structure a modified guanosine nucleotide
  • poly-A tail a sequence of up to 200 adenosine nucleotides
  • the mRNA coding for the tumour antigen or the nucleic acid molecule coding for the inhibitor of the herein defined vaccine/inhibitor combination may be modified, and thus stabilized, especially if the nucleic acid molecule is in the form of a coding nucleic acid, preferably an mRNA, by modifying the G/C content of the nucleic acid molecule, preferably of the coding region thereof.
  • the G/C content of the coding region of the nucleic acid molecule i.e. an mRNA coding for the tumour antigen or nucleic acid molecule coding for the inhibitor of the herein defined vaccine/inhibitor combination, is modified, particularly increased, compared to the G/C content of the coding region of its particular wild type coding sequence, e.g. the unmodified mRNA.
  • the encoded amino acid sequence of the nucleic acid sequence is preferably not modified compared to the coded amino acid sequence of the particular wild type mRNA.
  • the modified mRNA coding for the tumour antigen or the nucleic acid molecule coding for the inhibitor of the herein defined vaccine/inhibitor combination is in the form of an mRNA or codes for an mRNA
  • the coding region of the modified nucleic acid is preferably modified compared to the corresponding region of the wild type mRNA or coding sequence such that at least one codon of the wild type sequence which codes for a tRNA, which is relatively rare in the cell, is exchanged for a codon, which codes for a tRNA, which is relatively frequent in the cell and carries the same amino acid as the relatively rare tRNA.
  • the sequences of the nucleic acid molecule of the inventive polymeric carrier cargo complex is modified such that codons for which frequently occurring tRNAs are available are inserted.
  • all codons of the wild type sequence, which code for a tRNA which is relatively rare in the cell can in each case be exchanged for a codon which codes for a tRNA, which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA.
  • the mRNA coding for the tumour antigen or the nucleic acid sequence coding for the inhibitor of the vaccine/inhibitor combination is associated with a vehicle, transfection or complexation agent for increasing the transfection efficiency of the nucleic acid sequence.
  • cationic or polycationic compounds including protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAn
  • cationic peptides in this context are e.g. Arg7, Arg 8 , Arg9, H3R9, R9H3, H3R9H3, YSSR9SSY, (RKH) 4 , Y(RKH) 2 R, etc.
  • Further preferred cationic or polycationic compounds, which can be used as transfection agent may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethylene-imine (PEI), cationic lipids, e.g.
  • PEI polyethylene-imine
  • DOTMA [1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride
  • DMRIE di-C14-amidine
  • DOTIM DOTIM
  • SAINT DC-Chol
  • BGTC CTAP
  • DOPC DODAP
  • DOPE Dioleyl phosphatidylethanol-amine
  • DOSPA DODAB
  • DOIC DOIC
  • DMEPC DOGS: Dioctadecylamidoglicylspermin
  • DIMRI Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide
  • DOTAP dioleoyloxy-3-(trimethylammonio)propane
  • DC-6-14 O,O-ditetradecanoyl-N-( ⁇ -trimethylammonioacetyl)diethanolamine chloride
  • CLIP1 rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]
  • modified polyaminoacids such as ⁇ -aminoacid-polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
  • modified Amidoamines such as pAMAM (poly(amidoamine)), etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
  • polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), etc.
  • polyallylamine sugar backbone based polymers
  • the mRNA coding for the tumour antigen contained in the vaccine component of the vaccine/inhibitor combination is formulated as an immunostimulatory composition comprising a) an adjuvant component, comprising or consisting of at least one (m)RNA, complexed with a cationic or polycationic compound, preferably as defined herein, and b) at least one free mRNA, encoding the tumour antigen.
  • an adjuvant component comprising or consisting of at least one (m)RNA, complexed with a cationic or polycationic compound, preferably as defined herein, and b) at least one free mRNA, encoding the tumour antigen.
  • the vaccine/inhibitor combination is formulated with the vaccine component and the inhibitor together in the same composition, preferably for use as a vaccine.
  • the vaccine and the inhibitor of the vaccine/inhibitor combination are formulated separately in different compositions for use in the treatment of cancer, i.e. one composition comprising or representing the vaccine component containing an mRNA encoding at least one tumour antigen as defined herein, and one further composition (composition B) comprising the inhibitor as defined herein, the compositions preferably formulated as pharmaceutical compositions.
  • a composition comprising the inhibitor and/or the vaccine component may be formulated with or comprise a pharmaceutically acceptable carrier and/or vehicle.
  • a pharmaceutically acceptable carrier typically includes the liquid or non-liquid basis of a composition comprising the inhibitor and/or the vaccine of the vaccine/inhibitor combination. If the composition is provided in liquid form, the carrier will typically be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions.
  • the injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e.
  • the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects.
  • Reference media are e.g. liquids occurring in "in vivo" methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in “in vitro” methods, such as common buffers or liquids.
  • Such common buffers or liquids are known to a skilled person.
  • Ringer-Lactate solution is particularly preferred as a liquid basis for the mRNA encoding at least one tumour antigen comprised in the vaccine component or for the inhibitor of the vaccine/inhibitor combination, if the inhibitor is provided as nucleic acid sequence.
  • compatible solid or liquid fillers or diluents or encapsulating compounds which are suitable for administration to a patient to be treated, may be used as well for a composition comprising the inhibitor and/or the vaccine component of the vaccine/inhibitor combination.
  • compatible means that these constituents of the inventive pharmaceutical composition are capable of being mixed with the nucleic acid as defined herein in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive pharmaceutical composition under typical use conditions.
  • a composition comprising the inhibitor and the vaccine component of the vaccine/inhibitor combination may comprise an adjuvant.
  • an adjuvant may be understood as any compound, which is suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response.
  • the vaccine when administered, the vaccine preferably elicits an innate immune response due to the adjuvant, optionally contained therein.
  • an adjuvant may be selected from an adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal, e.g. an adjuvant protein as defined above or an adjuvant as defined in the following.
  • adjuvants suitable for depot and delivery are cationic or polycationic compounds as defined above for mRNA coding for the tumour antigen or for the inhibitor as vehicle, transfection or complexation agent.
  • an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal.
  • the adjuvant may be selected from the group consisting of, without being limited thereto, cationic or polycationic compounds as defined above, from chitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMERTM (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINETM (propanediamine); BAY R100
  • TM liposomes
  • LOXORIBINETM (7-allyl-8-oxoguanosine); LT 5 oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MF59TM; (squalenewater emulsion); MONTANIDE ISA 51 TM (purified incomplete Freund's adjuvant); MONTANIDE ISA 720TM (metabolisable oil adjuvant); MPLTM (3-Q-desacyl-4'-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt); MURAMETIDETM (Nac-Mur-L-Ala-D-Gln-OCH3);
  • composition comprising the inhibitor and the vaccine component of the vaccine/inhibitor combination can additionally contain one or more auxiliary substances in order to increase its immunogenicity or immunostimulatory capacity, if desired.
  • auxiliary substances A synergistic action of the vaccine component or an inhibitor as defined herein and of an auxiliary substance, which may be optionally contained in the vaccine component or may be formulated with the inhibitor, is preferably achieved thereby.
  • various mechanisms can come into consideration in this respect.
  • DCs dendritic cells
  • auxiliary substances for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class of suitable auxiliary substances.
  • auxiliary substance any agent that influences the immune system in the manner of a "danger signal" (LPS, GP96, etc.) or cytokines, such as GM-CSF, which allow an immune response to be enhanced and/or influenced in a targeted manner.
  • a "danger signal” LPS, GP96, etc.
  • cytokines such as GM-CSF
  • auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, that further promote the innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.
  • cytokines such as monokines, lymphokines, interleukins
  • a composition comprising the inhibitor and the vaccine component of the vaccine/inhibitor combination can also additionally contain any further compound, which is known to be immunostimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.
  • any further compound which is known to be immunostimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.
  • immunostimulating compounds are immunostimulatory nucleic acids which are known to bind to TLR receptors.
  • an immunostimulatory nucleic acid can be in the form of a(n) (immunostimulatory) CpG nucleic acid, in particular CpG-RNA or CpG-DNA, which preferably induces an innate immune response.
  • a CpG-RNA or CpG-DNA used according to the invention can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA).
  • the CpG nucleic acid used according to the invention is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA).
  • such CpG nucleic acids have a length as described above.
  • the CpG motifs are unmethylated.
  • an immunostimulatory nucleic acid as defined above may be in the form of an immunostimulatory RNA (isRNA), which preferably elicits an innate immune response.
  • an immunostimulatory RNA may be any (double-stranded or single-stranded) RNA, e.g. a coding RNA, as defined herein.
  • the immunostimulatory RNA may be a single-stranded, a double-stranded or a partially double-stranded RNA, more preferably a single-stranded RNA, and/or a circular or linear RNA, more preferably a linear RNA. More preferably, the immunostimulatory RNA may be a (linear) single-stranded RNA.
  • the immunostimulatory RNA may be a (long) (linear) single-stranded non-coding RNA.
  • the isRNA carries a triphosphate at its 5'-end which is the case for in vitro transcribed RNA.
  • An immunostimulatory RNA may also occur as a short RNA oligonucleotide as defined herein.
  • An immunostimulatory RNA as used herein may furthermore be selected from any class of RNA molecules, found in nature or being prepared synthetically, and which can induce an innate immune response and may support an adaptive immune response induced by an antigen. In this context, an immune response may occur in various ways.
  • T-lymphocytes are typically divided into two sub-populations, the T-helper 1 (Th1) cells and the T-helper 2 (Th2) cells, with which the immune system is capable of destroying intracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).
  • the two Th cell populations differ in the pattern of the effector proteins (cytokines) produced by them.
  • Th1 cells assist the cellular immune response by activation of macrophages and cytotoxic T-cells.
  • Th2 cells promote the humoral immune response by stimulation of B-cells for conversion into plasma cells and by formation of antibodies (e.g. against antigens).
  • the Th1/Th2 ratio is therefore of great importance in the induction and maintenance of an adaptive immune response.
  • the Th1/Th2 ratio of the (adaptive) immune response is preferably shifted in the direction towards the cellular response (Th1 response) and a cellular immune response is thereby induced.
  • the innate immune system which may support an adaptive immune response, may be activated by ligands of Toll-like receptors (TLRs).
  • TLRs are a family of highly conserved pattern recognition receptor (PRR) polypeptides that recognize pathogen-associated molecular patterns (PAMPs) and play a critical role in innate immunity in mammals.
  • PRR pattern recognition receptor
  • TLR1 - TLR13 Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13
  • TLR1 - TLR13 Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13
  • TLR1 - TLR13 Toll-like receptors: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7,
  • ligands for TLR9 include certain nucleic acid molecules and that certain types of RNA are immunostimulatory in a sequence-independent or sequence-dependent manner, wherein these various immunostimulatory RNAs may e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors such as RIG-1, MDA-5, etc. E.g. Lipford et al.
  • G,U-containing oligoribonucleotides as immunostimulatory by acting via TLR7 and TLR8 (see WO 03/086280 ).
  • the immunostimulatory G,U-containing oligoribonucleotides described by Lipford et al. were believed to be derivable from RNA sources including ribosomal RNA, transfer RNA, messenger RNA, and viral RNA.
  • the immunostimulatory RNA used as a further compound of the composition comprising the vaccine component of the vaccine/inhibitor combination or a composition comprising the inhibitor and the vaccine component of the vaccine/inhibitor combination, may thus comprise any RNA sequence known to be immunostimulatory, including, without being limited thereto, RNA sequences representing and/or encoding ligands of TLRs, preferably selected from human family members TLR1 - TLR10 or murine family members TLR1 - TLR13, more preferably selected from (human) family members TLR1 - TLR10, even more preferably from TLR7 and TLR8, ligands for intracellular receptors for RNA (such as RIG-1 or MDA-5, etc.) (see e.g.
  • immunostimulatory RNA molecules used as a further compound of the composition comprising the vaccine component of the vaccine/inhibitor combination or a composition comprising the inhibitor and the vaccine component of the vaccine/inhibitor combination, may include any other RNA capable of eliciting an immune response.
  • an immunostimulatory RNA may include ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), and viral RNA (vRNA).
  • Such an immunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30 nucleotides.
  • such immunostimulatory nucleic acid sequences consist of or comprise a nucleic acid of formula (I) or (II): G l X m G n (formula (I)) wherein:
  • such immunostimulatory nucleic acid sequences consist of or comprise a nucleic acid of formula (III) or (IV): (N u G l X m G n N v ) a (formula (III)) wherein:
  • the definition of bordering elements N u and N v is identical to the definitions given above for N u and N v .
  • emulsifiers such as, for example, Tween®
  • wetting agents such as, for example, sodium lauryl sulfate
  • colouring agents such as, for example, sodium lauryl sulfate
  • taste-imparting agents pharmaceutical carriers
  • tablet-forming agents such as, for example, stabilizers; antioxidants; preservatives.
  • composition comprising the vaccine component and/or the inhibitor of the vaccine/inhibitor combination may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or infusion techniques.
  • compositions comprising the vaccine component and/or the inhibitor of the vaccine/inhibitor combination are administered intradermally to reach APCs in the dermis.
  • composition comprising the vaccine component and/or the inhibitor of the vaccine/inhibitor combination as defined herein may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • composition comprising the vaccine component and/or the inhibitor of the vaccine/inhibitor combination may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the composition comprising the vaccine component and/or the inhibitor of the vaccine/inhibitor combination may be formulated in a suitable ointment, containing the vaccine component and/or the inhibitor and optionally further compounds as defined herein suspended or dissolved in one or more carriers.
  • composition comprising the vaccine component and/or the inhibitor typically comprise a "safe and effective amount" of the components of the vaccine/inhibitor combination.
  • a "safe and effective amount” preferably means an amount of the vaccine component and/or the inhibitor of the vaccine/inhibitor combination as defined herein that is sufficient to significantly induce a positive modification of a disease or disorder as defined herein.
  • a "safe and effective amount” is small enough to avoid serious side-effects and to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.
  • a pharmaceutical composition comprises as vaccine component an mRNA encoding at least one tumour antigen and an inhibitor of MHC class I restricted antigen presentation, both preferably as defined above.
  • the pharmaceutical composition is preferably formulated and administered as defined above for the components of the vaccine/inhibitor combination.
  • the combination of the vaccine component and the inhibitor of MHC class I restricted antigen presentation as defined according to the present invention may occur either as one composition, e.g. as a kit, comprising all these components in one and the same mixture, or may occur in more than one composition, e.g. as a kit of parts, wherein the different components form different parts of such kit of parts.
  • These different components may be formulated each as a vaccine, a pharmaceutical composition or as a composition as defined above.
  • each of the different parts of the kit comprises a different component, e.g. one part comprising the vaccine component as defined herein, one further part comprising the inhibitor of MHC class I restricted antigen presentation as defined herein, etc.
  • a kit or a kit of parts may further comprise any ingredient as defined above for the vaccine/inhibitor combination.
  • the TEIPP-specific CD8+-mediated immune response by utilizing the vaccine/inhibitor combination may be additionally improved by an additional vaccine added to the pharmaceutical composition(s) containing the inhibitor as described herein.
  • an additional vaccine added to the pharmaceutical composition(s) containing the inhibitor as described herein is possible, because TEIPPs of the antigen(s) contained in the vaccine can be presented by the APCs additionally to the TEIPPs of housekeeping genes and increase the probability that tumour cells, which express the antigen(s) and present TEIPPs generated from these antigens, are recognized and eliminated by the immune system.
  • the vaccine/inhibitor combination is combined with an additional vaccine.
  • the vaccine/inhibitor combination may be formulated as two separated compositions as defined above, wherein a first composition preferably comprises the vaccine component as defined above of the vaccine/inhibitor combination and the second composition preferably comprises the inhibitor of the vaccine/inhibitor combination and preferably an additional vaccine.
  • this second pharmaceutical composition may also be split into two separate compositions. These compositions are preferably prepared as defined above, e.g. as pharmaceutical compositions.
  • the inhibitor and the additional vaccine are to be administered at the same time and/or the same administration site to ensure that the inhibitor and the additional vaccine reach the same cells.
  • the inhibitor and the additional vaccine may be administered at the same time and/or the same administration site.
  • Such an additional vaccine may be as defined above for the vaccine of vaccine/inhibitor combination.
  • the first composition and the second composition are preferably administered separated in time or locally separated - similarly to what has already been outlined above for the vaccine/inhibitor combination alone.
  • the second composition comprising the inhibitor and the additional vaccine may be administered, e.g. prior, concurrent or subsequent to the first composition, comprising the vaccine component of the vaccine/inhibitor combination, or vice versa.
  • the first composition and the second composition may be administered at different administration sites, or at the same administration site, preferably, when administered in a time staggered manner.
  • first composition comprising the vaccine component of the vaccine/inhibitor combination
  • second composition as defined above comprising the inhibitor of the vaccine/inhibitor combination and, optionally, the additional vaccine (either as one or as two separate compositions B and C) to induce an immune response against escaped tumour cells, which have lost presentation of antigen epitopes on MHC class I molecules.
  • the vaccine/inhibitor combination or the inventive pharmaceutical composition comprising a vaccine component and an inhibitor and, optionally, an additional vaccine (preferably in form of a first and a second composition as described above) may be used for human and also for veterinary medical purposes, preferably for human medical purposes.
  • the present invention is also directed to the first medical use of the vaccine/inhibitor combination or the inventive pharmaceutical composition and, optionally, an additional vaccine as defined herein (preferably in form of a first and a second composition as described above) as a medicament or as a vaccine.
  • the present invention is also directed to the second medical use of the vaccine/inhibitor combination or the inventive pharmaceutical composition and, optionally, an additional vaccine as defined herein (preferably in form of a first and a second composition as described above) for the treatment of diseases as defined herein, or of kits comprising the same for the preparation of a medicament for the treatment and/or amelioration of various diseases as defined herein.
  • diseases as mentioned herein are selected from cancer or tumour diseases, which are associated with a loss in MHC class I restricted antigen presentation.
  • Cancer or tumour diseases disclosed herein include e.g. colon carcinomas, melanomas, renal carcinomas, lymphomas, acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), gastrointestinal tumours, pulmonary carcinomas, gliomas, thyroid tumours, mammary carcinomas, prostate tumours, hepatomas, various virus-induced tumours such as, for example, papilloma virus-induced carcinomas (e.g. cervical carcinoma), adenocarcinomas, herpes virus-induced tumours (e.g.
  • Burkitt's lymphoma EBV-induced B-cell lymphoma), heptatitis B-induced tumours (hepatocell carcinoma), HTLV-1- and HTLV-2-induced lymphomas, acoustic neuromas/neurinomas, cervical cancer, lung cancer, pharyngeal cancer, anal carcinomas, glioblastomas, lymphomas, rectal carcinomas, astrocytomas, brain tumours, stomach cancer, retinoblastomas, basaliomas, brain metastases, medulloblastomas, vaginal cancer, pancreatic cancer, testicular cancer, melanomas, thyroidal carcinomas, bladder cancer, Hodgkin's syndrome, meningiomas, Schneeberger disease, bronchial carcinomas, hypophysis tumour, Mycosis fungoides, oesophageal cancer, breast cancer, carcinoids, neurinomas, spinaliomas, Burkitt's lymphomas, larynge
  • kits of parts as defined by claim 15 and as already defined herein in the context of pharmaceutical compositions typically comprise as components alone or in combination with further components as defined herein at least one vaccine component comprising an mRNA encoding at least one tumour antigen and in a different part of the kit, at least one inhibitor of MHC class I restricted antigen presentation as defined herein.
  • At least one part of the kit comprises at least one vaccine component as defined herein, and at least one further part of the kit at least one inhibitor of MHC class I restricted antigen presentation as defined herein, and, optionally, at least one further part of the kit an additional vaccine as described herein.
  • the kit or kit of parts may furthermore contain technical instructions with information on the administration and dosage of the vaccine/inhibitor combination, e.g. any of its components or parts, e.g. if the kit is prepared as a kit of parts.
  • DNA sequences encoding Gallus gallus ovalbumin mRNA (R1435) and TAP inhibitor UL49.5 mRNA (R1617) were prepared and used for subsequent in vitro transcription reactions.
  • control RNA mRNA coding for Photinus pyralis luciferase was used (R1533 and R491).
  • the DNA sequences coding for the above mentioned mRNAs were prepared.
  • the constructs were prepared by modifying the wild type coding sequences by introducing a GC-optimized sequence for a better codon usage and stabilization, stabilizing sequences derived from alpha-globin-3'-UTR (muag: (mutated alpha-globin-3'-UTR)), a stretch of 70 x adenosine at the 3'-terminal end (poly-A-tail), a stretch of 30 x cytidine at the 3'- terminal end (poly-C-tail).
  • alpha-globin-3'-UTR alpha-globin-3'-UTR
  • a stretch of 70 x adenosine at the 3'-terminal end poly-A-tail
  • a stretch of 30 x cytidine at the 3'- terminal end (poly-C-tail).
  • the respective DNA plasmids prepared according to Example 1 were transcribed in vitro using T7 polymerase. Subsequently the mRNAs were purified using PureMessenger® (CureVac, Tübingen, Germany).
  • the mRNA used as adjuvant component of the vaccine in the experiments below was complexed with protamine by addition of protamine to the mRNA in the ratio (1:2) (w/w). After incubation for 10 min, the same amount of free mRNA used as antigen was added.
  • R1435 adjuvant component consisting of mRNA coding for Gallus gallus ovalbumine according to SEQ ID NO. 1 complexed with protamine in a ratio of 2:1 (w/w) and free mRNA coding for Gallus gallus ovalbumine (antigen mRNA) according to SEQ ID NO. 1 (ratio 1:1; complexed RNA:free RNA).
  • control vaccine adjuvant component consisting of mRNA coding for Photinus pyrialis luciferase according to SEQ ID NO. 2 complexed with protamine in a ratio of 2:1 (w/w) and free mRNA coding for Photinus pyralis luciferase (antigen mRNA) according to SEQ ID NO 2 (ratio 1:1; complexed RNA:free RNA).
  • mice On the indicated days of the respective experiments C57BL/6 mice (8 mice per group) were vaccinated intradermally with the mRNA vaccines R1435 or R1533 (coding for Gallus gallus ovalbumine or Photinus pyralis luciferase as control, respectively). Injection was performed with 64 ⁇ g (experiment 1 ( Fig. 1 )) or 32 ⁇ g (experiment 2 ( Fig. 2 )) mRNA/mouse/day or Ringer-lactate as buffer control (RiLa).
  • mice were challenged subcutaneously (right flank) with 1x10 6 (experiment 1 ( Fig. 1 )) or 0.3x10 6 (experiment 2 ( Fig. 2 )) E.G7-OVA cells per mouse (volume 100 ⁇ l in PBS).
  • mice were intradermally injected with the mRNAs R1617 or R491 (coding for the TAP-inhibitor UL49.5 or Photinus pyralis luciferase as control respectively).
  • mRNAs R1617 or R491 coding for the TAP-inhibitor UL49.5 or Photinus pyralis luciferase as control respectively.

Claims (18)

  1. Composition comprenant
    un ARNm codant pour au moins un antigène tumoral, son variant antigénique tumoral ayant au moins 80 % d'identité de séquence vis-à-vis de la séquence d'acides aminés de type sauvage dudit au moins un antigène tumoral ou un fragment de 6 à 20 acides aminés contigus dudit au moins un antigène tumoral, où le fragment comprend son épitope antigénique de cellules T, et
    au moins un inhibiteur de présentation à l'antigène restreint au CMH de classe I, où ledit au moins un inhibiteur est un inhibiteur du transporteur associé à la présentation antigénique (TAP) sélectionné dans le groupe constitué de US6 du HCMV, de ICP47 du virus de l'herpès simplex, de UL49.5 du virus de la varicelle, et de BNLF2a du virus d'Epstein-Barr, ou un variant fonctionnel inhibiteur de TAP de n'importe lesquels des inhibiteurs ci-dessus ayant au moins 80 % d'identité de séquence vis-à-vis de la séquence d'acides aminés de type sauvage de l'un quelconque desdits inhibiteurs, ou un acide nucléique codant pour l'un quelconque desdits inhibiteurs ou ses variants fonctionnels inhibiteurs de TAP.
  2. Composition selon la revendication 1, dans laquelle l'inhibiteur est codé par un acide nucléique.
  3. Composition selon la revendication 2, dans laquelle l'inhibiteur est codé par un ARNm.
  4. Composition selon l'une quelconque des revendications 1 à 3, la composition ne comprenant pas de chaîne lourde du CMH de classe I, ni/ou de protéine β2m, ni/ou de protéine de fusion comprenant une chaîne lourde du CMH de classe I ou de protéine β2m, ni/ou d'acide nucléique ou de vecteur codant pour une quelconque protéine ou protéine de fusion.
  5. Composition selon l'une quelconque des revendications 1 à 4, la composition ne comprenant pas de cellule de présentation à l'antigène, par exemple de cellule dendritique.
  6. Composition selon l'une des revendications 1 à 5, la composition comprenant en outre un support et/ou un véhicule pharmaceutiquement acceptable.
  7. Composition selon l'une quelconque des revendications 1 à 6, la composition comprenant en outre un adjuvant, un antigène tumoral et/ou un acide nucléique additionnel codant pour un antigène tumoral.
  8. Composition selon l'une des revendications 1 à 7 pour l'utilisation comme médicament.
  9. Composition selon l'une des revendications 1 à 7 pour l'utilisation dans le traitement du cancer.
  10. Première composition comprenant un ARNm codant pour au moins un antigène tumoral, son variant antigénique tumoral ayant au moins 80 % d'identité de séquence vis-à-vis de la séquence d'acides aminés de type sauvage dudit au moins un antigène tumoral ou un fragment de 6 à 20 acides aminés contigus dudit au moins un antigène tumoral, le fragment comprenant son épitope antigénique de cellules T,
    pour l'utilisation dans un procédé de traitement du cancer,
    le procédé de traitement comprenant également l'administration d'une seconde composition comprenant au moins un inhibiteur de présentation à l'antigène restreint au CMH de classe I, ledit au moins un inhibiteur étant un inhibiteur du transporteur associé à la présentation antigénique (TAP), préférablement un ARNm codant pour ledit au moins un inhibiteur, sélectionné dans le groupe constitué de US6 du HCMV, de ICP47 du virus de l'herpès simplex, de UL49.5 du virus de la varicelle, et de BNLF2a du virus d'Epstein-Barr ou un variant fonctionnel inhibiteur de TAP de n'importe lesquels des inhibiteurs ci-dessus ayant au moins 80 % d'identité de séquence vis-à-vis de la séquence d'acides aminés de type sauvage de l'un quelconque desdits inhibiteurs et
    le procédé de traitement, éventuellement, comprenant en outre l'administration d'une troisième composition comprenant un antigène tumoral additionnel et/ou un acide nucléique codant pour un antigène tumoral additionnel.
  11. Composition pour l'utilisation selon la revendication 10, dans laquelle lesdites première et seconde composition sont administrées au·à la patient·e d'une manière étalée dans le temps.
  12. Composition pour l'utilisation selon l'une des revendications 10 ou 11, dans laquelle lesdites première et seconde composition sont administrées au·à la patient·e à différents lieux d'administration.
  13. Composition pour l'utilisation selon l'une des revendications 10 à 12, dans laquelle la troisième composition est administrée en même temps et/ou au même lieu d'administration que la seconde composition.
  14. Composition pour l'utilisation selon l'une des revendications 10 et 12, dans laquelle la troisième et la seconde composition sont administrées avant, simultanément ou ultérieurement à la première composition.
  15. Kit de parties comprenant
    comme première partie une première composition comprenant un ARNm codant pour au moins un antigène tumoral, son variant antigénique tumoral ayant au moins 80 % d'identité de séquence vis-à-vis de la séquence d'acides aminés de type sauvage dudit au moins un antigène tumoral ou un fragment de 6 à 20 acides aminés contigus dudit au moins un antigène tumoral, le fragment comprenant son épitope antigénique de cellules T, et
    comme seconde partie une seconde composition comprenant au moins un inhibiteur de présentation à l'antigène restreint au CMH de classe I, ledit au moins un inhibiteur étant un inhibiteur de TAP sélectionné dans le groupe constitué de US6 du HCMV, de ICP47 du virus de l'herpès simplex, de UL49.5 du virus de la varicelle, et de BNLF2a du virus d'Epstein-Barr, ou un variant fonctionnel inhibiteur de TAP de n'importe lesquels des inhibiteurs ci-dessus ayant au moins 80 % d'identité de séquence vis-à-vis de la séquence d'acides aminés de type sauvage de l'un quelconque desdits inhibiteurs, ou un acide nucléique codant pour l'un quelconque desdits inhibiteurs ou ses variants fonctionnels inhibiteurs de TAP.
  16. Kit de parties selon la revendication 15, l'inhibiteur étant codé par un ARNm.
  17. Kit de parties selon la revendication 15 ou 16 comprenant additionnellement comme troisième partie une troisième composition, comprenant un antigène tumoral et/ou un acide nucléique additionnel codant pour un antigène tumoral.
  18. Kit de parties selon l'une des revendications 15, 16 ou 17 pour l'utilisation dans le traitement du cancer.
EP11808600.8A 2010-12-29 2011-12-27 Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh Active EP2658569B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP11808600.8A EP2658569B1 (fr) 2010-12-29 2011-12-27 Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
PCT/EP2010/007969 WO2012089225A1 (fr) 2010-12-29 2010-12-29 Combinaison de vaccination et d'inhibition de la présentation des antigènes restreinte par le cmh de classe i
EP11808600.8A EP2658569B1 (fr) 2010-12-29 2011-12-27 Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh
PCT/EP2011/006585 WO2012089338A1 (fr) 2010-12-29 2011-12-27 Combinaison de vaccination et d'inhibition de la présentation d'antigène restreinte à une classe de cmh

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CN110177544A (zh) 2016-11-29 2019-08-27 普尔泰克健康有限公司 用于递送治疗剂的外泌体

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